The mature nervous system contains thousands of distinct neuronal subtypes, each with specialized identity, connectivity and function within neural circuits. How this remarkable diversity is generated during development is poorly understood. Understanding the process of neuronal subtype diversification will yield important insights into how neural circuits are assembled to produce distinct behaviors, as well as reveal why specific neuronal subtypes are selectively vulnerable in some neurodegenerative diseases. In this proposal, I will focus on the subtype diversification of V1 interneurons (IN), a class of inhibitor neurons in the vertebrate spinal cord that are essential for controlling motor circuit activity. During spinal cord development, the V1 progenitor domain produces more than two dozen distinct V1 IN subtypes, raising the important question of how diverse IN cell types are derived from the same progenitor domain. One of the best-studied V1 IN subtypes is the Renshaw cell (RC), which provides recurrent inhibition of motor neurons (MN). RCs are a cell type of special interest given evidence of selective impairment of the RC recurrent inhibitory circuit in the MN disease amyotrophic lateral sclerosis (ALS). However, molecular mechanisms controlling differentiation of V1 progenitors into specialized subtypes such as RCs are currently unknown. Our lab has pioneered the use of embryonic stem cell (ESC)-derived neurons for studying molecular mechanisms of spinal MN differentiation and subtype diversification. In preliminary studies, I developed and optimized differentiation of ESCs to V1 INs, including showing that they recapitulate normal V1 IN development. Importantly, in vitro-derived V1 INs express V1 subtype-specific molecular markers, including the RC-specific marker calbindin (Cb). In this project proposal, I will take advantage of the in vitro differentiation system to test the specific hypothesis that specification of different V1 subtypes, including RCs, is dependent on timing of neurogenesis in a manner regulated by the Notch signaling pathway. I anticipate that these results will (1) establish an experimentally accessible in vitro model of RCs that can be used to study their role in normal spinal physiology and in neurological diseases such as ALS; and (2) provide unprecedented insights into molecular mechanisms generating V1 subtype diversity, knowledge that can be used to efficiently differentiate pluripotent stem cells into clinically-relevant cell types for modeling disease, drug discovery, and cellular replacement therapy.